Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/42—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins
  • C07K16/4208—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
  • C07K16/4241—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man

Definitions

• the invention relates to human, monoclonal antibodies which react with islet cells of the pancreas, their use as standard in the determination of antibodies against an islet cell antigen, and a method for the determination of antibodies against an islet cell antigen of the pancreas.
• Antibodies that react with the endocrine cells of the pancreas, the islet cells, are often found in sera from prediabetic or newly diagnosed diabetics. These antibodies are commonly referred to as islet cell antibodies (ICA).
• ICA islet cell antibodies
• the typical ICA are of the IgG isotype (G. Botazzo et al., Lancet 2 (1974) 1279-1283). They react selectively with the endocrine cells of the pancreas, but not with the exocrine cells, the execution ducts or the connective tissue.
• the likewise detectable autoreactive antibodies of the IgM isotype do not show any preferential islet cell staining, but also react in the same way with other tissues, such as the pituitary, thyroid, T-lymphocytes and adrenal glands (E. Garzelli et al., J. Clin. Invest. 77 , 1986, 1627-1631; S. Srikanta et al., Mol. Biol. Med. 3, 1986, 113-127). They probably belong to the type of low-affinity natural autoantibodies of low specificity, which also occur in the blood of healthy people (Seigneurin et al., Blood 71 (1988), 581).
• the presence of the ICA from the IgG isotype in the serum is a predictive marker for the so-called insulin-dependent diabetes (synonymous with juvenile diabetes or type I diabetes). These autoantibodies can be detected in the serum up to nine years before the onset of diabetes.
• islet cell antigens which are, however, not specific for islet cells.
• the antigens or epitopes of these antigens are also found in other endocrine tissues such as the pituitary, thyroid, parathyroid, thymus, adrenal glands as well as in melanocytes.
• GAD glutamate decarboxylase
• human monoclonal antibodies of the IgG isotype against human pancreatic islet cells obtainable by immortalizing human lymphocytes from prediabetic or diabetic patients, treating the culture supernatant of the immortalized cells with a conjugate of antibodies against human Fc ⁇ and labeling, followed by treatment with human immunoglobulin, incubating with immobilized human pancreatic islet cells or immobilized GAD, identifying an immortalized human cell culture which produces an antibody against pancreatic islet cells by determining the label bound to the immobilized islet cells or the immobilized GAD, isolating a human immortalized cell, producing this antibody, multiplying this immortalized cell and isolating the monoclonal antibody produced by these cells.
• the monoclonal antibodies obtainable according to the invention react specifically with purified GAD. After incubation with various tissue sections, the antibodies according to the invention react with islet cells of the pancreas and the cerebellum, but not with other tissues such as e.g. B. from the adrenal gland, stomach, intestine, pituitary gland, cerebral cortex, lungs, liver or thyroid.
• the binding of the antibodies according to the invention to pancreatic sections can be weakened or prevented entirely by preincubation with at least one of three diabetic sera. This shows that the antibodies according to the invention are relevant not only for the diabetes of the particular donor from whose blood they were obtained, but generally for type I diabetes mellitus.
• Epstein-Barr virus EBV
• other methods known to those skilled in the art e.g. hybridoma technology can also be used.
• a conjugate of antibodies against human Fc ⁇ and a label is used to detect immortalized cells that produce the desired antibody.
• a conjugate of human Fc ⁇ -binding Fab fragments and a label is preferred here, as is obtainable by the method of Ishikawa et.al., J. Immunoassay 4 (1983), page 209.
• Fc ⁇ -binding Fab fragments both monovalent Fab or F (ab ') fragments and divalent F (ab') 2 fragments can be used.
• the labels familiar to the person skilled in the art can be used as the label, usually an enzyme, a fluorescent or chemiluminescent dye or a radioactive isotope is used.
• the conjugate of antibodies against human Fc ⁇ and a label is first incubated with the culture supernatant of the immortalized cells, then treated with human immunoglobulin and finally incubated with immobilized human pancreatic islet cells or immobilized GAD.
• Pancreatic tissue sections are preferably used as immobilized islet cells.
• the immortalized cell line producing an antibody against pancreatic islet cells is then identified by determining the label bound to the immobilized islet cells or the immobilized GAD. Depending on the type of labeling, this determination is carried out via an appropriate enzyme reaction or via measurement of fluorescence or chemiluminescence.
• the cells whose culture supernatant shows a positive reaction are separated. This is preferably done using a fluorescence-activated cell sorter.
• the cell clones isolated in this way are propagated and those cell clones which produce antibodies against pancreatic islet cells are identified as described above.
• the antibody is then isolated from the culture supernatant of these cell clones by methods familiar to the person skilled in the art.
• a preferred subject of the invention are monoclonal antibodies which are capable of binding to human pancreatic islet cells or to glutamate decarboxylase in an equivalent manner to the antibodies produced by the cell lines ECACC 90121401, ECACC 90121402, ECACC 90121403 or DSM ACC 2017.
• antibody capable of binding in an equivalent manner means antibodies in which an epitope overlap with the defined, known antibody is detectable. This epitope overlap can easily be detected using a competitive test system. To do this, an enzyme immunoassay is used to check the extent to which an antibody competes with the known antibody for binding to a defined antigen or a special epitope. To this end, immobilized islet cells, for example pancreatic sections, are incubated with the known monoclonal antibody in labeled form and an excess of the considered drawn antibody. By detecting the bound label, it can then easily be determined to what extent the antibody under consideration can displace the defined antibody from the binding. If there is a displacement of at least 50% with a 105-fold excess, there is an epitope overlap.
• the antibodies obtainable from the cell lines ECACC 90121401, ECACC 90121402, ECACC 90121403 or DSM ACC 2017 are particularly preferred.
• the invention further relates to the cell lines ECACC 90121401, ECACC 90121402, ECACC 90121403 and DSM ACC 2017.
• Another object of the invention is a method for producing human monoclonal antibodies of the IgG isotype against human pancreatic islet cells by immortalizing human lymphocytes from prediabetic or diabetic patients, treating the culture supernatant of the immortalized cells with a conjugate of antibodies against human Fc ⁇ and a label, subsequent treatment with human immunoglobulin, incubation with immobilized human pancreatic islet cells or immobilized GAD, identifying an immortalized human cell culture which produces an antibody against pancreatic islet cells, by determining the label bound to the immobilized islet cells or the immobilized GAD, isolating a human immortalized cell producing this antibody, multiplying this immortalized cell and isolating the monoclonal antibody produced by these cells.
• Another object of the invention is therefore a process for the production of monoclonal antibodies against epitopes of an islet cell antigen that are not recognized by known monoclonal antibodies against an islet cell antigen, in which one first produces monoclonal antibodies against an islet cell antigen according to the process of the invention, these in the presence of incubated labeled known monoclonal antibodies against an islet cell antigen with immobilized islet cells and, after detection of the bound label, selects those antibodies which can displace the known antibody from binding.
• Another object of the invention is a method for obtaining the islet cell antigen which binds to the antibodies produced by the cell lines ECACC 90121401, ECACC 90121402, ECACC 90121403 and / or DSM ACC 2017 by lysing human or animal pancreatic islet cells, pancreatic homogenates, or brain homogenates, in particular Cerebellar homogenates, separation of insoluble material, treatment of the solution with an immobilized monoclonal antibody obtainable from the cell lines ECACC 90121401, ECACC 90121402, ECACC 90121403 and / or DSM ACC 2017 or an antibody which binds to human pancreas islet cells in an equivalent manner and recovery of the to the antibody-binding islet cell antigen.
• the animal pancreatic islet cells, pancreatic homogenates or brain homogenates can e.g. B. from the corresponding organs of pork, beef, rats or a mouse.
• the antibodies are preferably immobilized by binding to a gel material such as e.g. Protein A Sepharose CL-4B.
• a gel material such as e.g. Protein A Sepharose CL-4B.
• the soluble fraction of the above-mentioned tissues obtained after centrifugation is applied to this immunoadsorbent after lysis and non-binding material is washed out.
• the binding antigen is then eluted from the immunoadsorbent. Elution is preferably carried out using 0.05 mol / 1 diethylamine pH 11.5, 0.5% sodium deoxycholate.
• the invention furthermore relates to anti-idiotypic antibodies which are directed against antibodies which react with an islet cell antigen, obtainable by immunization with an antibody according to the invention against an islet cell antigen and isolation of the desired antibodies from the serum of the immunized animals by affinity chromatography. Immunization is carried out in the animals commonly used for this, e.g. Sheep or rabbit.
• Another preferred object of the invention are monoclonal anti-idiotypic antibodies, obtainable by immunization with an antibody according to the invention against an islet cell antigen, immortalization of the spleen cells of the immunized animals, cloning of such immortalized cells which produce antibodies which bind to antibodies against islet cells of the pancreas and Isolation of the antibodies produced by these clones by known methods.
• the culture supernatant of the immortalized cells is incubated with an immobilized antibody against an islet cell antigen.
• an immobilized antibody against an islet cell antigen For the detection of bound anti-idiotypic antibody from the culture supernatant, a conjugate of a label and antibodies against Fc ⁇ of the animal species from which the immortalized cells were obtained is used.
• a conjugate of Fc ⁇ -binding Fab fragments and a label is preferred here, as is the case according to the method of Ishikawa et.al. J. Immunoassay 4 (1983), 209.
• Fc ⁇ -binding Fab fragments both monovalent Fab or F (ab ') fragments and divalent F (ab') 2 fragments can be used.
• the labels familiar to the person skilled in the art can be used as the label; an enzyme, a fluorescence or chemiluminescent dye or a radioactive isotope is usually used.
• Another object of the invention is a method for producing anti-idiotypic antibodies by immunization with an antibody according to the invention against an islet cell antigen and isolation of the anti-idiotypic antibody from the serum of the immunized animals by affinity chromatography.
• Another preferred object of the invention is a method for the production of monoclonal anti-idiotypic antibodies by immunization with an antibody according to the invention against an islet cell antigen, immortalization of the spleen cells of the immunized animals, cloning of such immortalized cells that produce antibodies that produce antibodies to antibodies against islet cells of the Pancreas bind and isolation of the antibodies produced by these clones by known methods.
• the invention further relates to a method for the determination of antibodies against an islet cell antigen by incubating the analysis material to be examined with a first receptor immobilized before or during the detection reaction and detection of the bound antibody contained in the analysis material via a labeled, second receptor binds this antibody.
• the culture supernatant of antibody-producing cells an isolated antibody or a body fluid, preferably subject serum, can be used as analysis material.
• the first receptor used for the detection of the antibodies in the analysis material can be immobilized in test tubes or on microtiter plates.
• a complete islet cell antigen preferably the complete natural GAD antigen, and the epitope responsible for binding can be used as a fragment of this antigen.
• an anti-idiotypic antibody which bears the internal image of the corresponding epitope is used as the immobilized first receptor.
• the first receptor is preferably biotinylated and then bound to a solid phase coated with streptavidin.
• the first receptor can also be used in the form of a tissue section, preferably pancreatic tissue, pancreatic islets or pancreatic islet cells.
• the labeled islet antigen GAD or preferably a labeled antibody against human immunoglobulin, preferably against human Fc ⁇ , is used as the labeled second receptor. All marking methods familiar to the person skilled in the art can be used as the marking.
• the detection of the bound sample antibody is carried out after separation of the liquid and solid phase by determining the marking in the isolated liquid or solid phase.
• the antibody is quantified in the analysis material by comparing the measured value with the value contained for a standard antibody of known concentration.
• a human monoclonal antibody according to the invention against human pancreatic islet cells is preferably used as the standard antibody. This can cause disturbances in the determination, e.g. when using murine antibodies caused by antibodies against mouse immunoglobulins in the patient's serum can be avoided.
• Another object of the invention is therefore the use of the human monoclonal antibodies according to the invention as a standard for the determination of antibodies against an islet cell antigen in the analysis material.
• the invention further relates to a method for the determination of antibodies against an islet cell antigen by incubating the analysis material to be examined with a first receptor immobilized before or during the detection reaction and detection of the bound antibody contained in the analysis material via a labeled, second receptor binds this antibody, the amount of the antibody contained in the analysis material being determined by
• the cell lines UH33 / 139.464 (ECACC 90121401), IID9-402 (ECACC 90121402) and AH25 / 43-116 (ECACC 90121403) according to the invention were obtained on December 14, 1990 at ECACC, Public Health Laboratory Service, Porton Down, Salisbury, Wiltshire SP 5 OJG, United Kingdom.
• the cell line MAK ⁇ GAD ⁇ H-HaE 12 was deposited on August 21, 1991 with the German Collection of Cell Cultures and Microorganisms GmbH, Mascheroderweg 1 b, D-3300 Braunschweig, under the number DSM ACC 2017.
• lymphocytes Only those lymphocyte donors whose sera gave a positive reaction when diluted at least or equal to 1:64 were used for the isolation of lymphocytes.
• the mononuclear cells in RPMI 1640 medium in a cell density of 2x106 cells / ml with 1 ml of the culture supernatant containing the EBV of the B 95-8 Marmoset cell line (ATCC CRL 1612) for 2 hours at 37 ° C and 5% C02 incubated.
• the cells were then washed and 2x104 cells per well of a 96-well microtiter plate were sown. At the beginning of the cultivation, 0.1% phytohaemagglutinin (Gibco, USA) was added to the medium.
• 25 ⁇ l of the culture supernatant was first mixed with a conjugate of human Fc ⁇ -binding Fab fragments and peroxidase (25 ⁇ l, 20 U / ml).
• This conjugate was prepared according to the method of Ishikawa et al., J. Immunoassay 4 (1983) 209. After incubation at 37 ° C for one hour, half the volume of 10% normal human serum was added and incubated again at 37 ° C for one hour. This mixture was applied to the pancreatic sections and incubated for two hours at 4 ° C.
• the immunohistochemical staining was carried out by incubation with 0.02% aminoethylcarbazole and 0.01% H2O2 for 15-25 minutes at room temperature .
• the evaluation was carried out under the light microscope. Those EBV lines whose culture supernatant gave a positive reaction (staining of the islet cells) were cloned.
• the cells were individually placed in 96-well microtiter plates using a fluorescence-activated cell sorter and fed with irradiated peripheral blood lymphocytes (5 ⁇ 104 cells / well, 4000 rad).
• the lymphocyte clones obtained in this way are cultivated in Iscove's modified Dulbeccos's complete medium (Gibco, with 15% FCS).
• the antibodies are removed from the supernatant by ammonium sulfate precipitation and affinity chromatography, e.g. B. isolated over protein A or protein G Sepharose.
• a competitive enzyme immunoassay was carried out to demonstrate the epitope overlap of an antibody with the monoclonal antibody ECACC 90121401, ECACC 90121402, ECACC 90121403 or DSM ACC 2017.
• the antibody to be assessed was incubated in various dilution stages for 1 hour at room temperature with human pancreatic sections and washed 3 ⁇ 5 minutes in PBS. Subsequently, peroxidase-labeled immune complexes from the abovementioned monoclonal antibodies with a conjugate of human Fc ⁇ -binding Fab fragments and peroxidase (preparation as described in Example 2) are added to the pancreatic sections and incubated for 2 hours at 4 ° C. After washing again (3 ⁇ 5 min.
• the detection of bound peroxidase-labeled antibody ECACC 90121401, ECACC 90121402, ECACC 90121403 or DSM ACC 2017 is carried out by immunohistochemical staining with aminoethyl carbazole (as described in Example 2).
• the staining obtained was compared to the staining obtained when incubated with the monoclonal antibody ECACC 90121401, ECACC 90121402, ECACC 90121403 or DSM ACC 2017 alone.
• Protein A - Sepharose CL-4B is in 0.1 mol / l borate buffer pH 8.2 with the human monoclonal antibody (ECACC 90121401, ECACC 90121402, ECACC 90121403 or DSM ACC 2017; 1 ml Sepharose binds 20 mg antibody) for at room temperature shaken gently for an hour. The Sepharose is then washed protein-free with 0.1 mol / l borate buffer pH 8.2 and filled into a column. The column material is mixed with 0.15 mol / l NaCl; 0.01 mol / l Tris-HCl pH 8.2; 1 mmol / l EDTA; 0.5% Triton X-100 equilibrated.
• the human monoclonal antibody ECACC 90121401, ECACC 90121402, ECACC 90121403 or DSM ACC 2017; 1 ml Sepharose binds 20 mg antibody
• the antigen bound to the immunoadsorbent is eluted using 0.05 mol / l diethylamine pH 11.5, 0.5% sodium deoxycholate.
• An aliquot of the column eluate is adsorptively bound to a solid phase (nitrocellulose or microtiter plate) and with the solution of an isolated from the cell lines ECACC 90121401, ECACC 90121402, ECACC 90121403 or DSM ACC 2017 of the produced islet cell antibody (1 ⁇ g / ml) for 2 hours incubated at room temperature and then washed with 3 x 350 ul 0.15 mol / l NaCl / 0.05% Tween 20.
• the positively reacting fractions are combined, neutralized by adding 0.5 mol / l NaH2PO4 and then against 0.15 mol / l NaCl; 0.01 mol / l Tris-HCl, pH 8.2; 1 mmol / l EDTA; 0.05% Triton X-100 dialyzed.
• the first receptor eg islet cell antigen GAD
• GAD islet cell antigen GAD
• D-biotinyl- ⁇ -amidocaproic acid-N-hydroxysuccinimide ester Boehringer Mannheim GmbH, Cat. No. 1008 960
• DBB diazo-para-aminobenzoylbiocytin
• Subject serum is diluted 1:25 with PBS containing 0.5% BSA (bovine serum albumin) and 100 ⁇ l of this dilution is pipetted into the receptor immobilized according to a).
• 100 ⁇ l of an isolated islet cell antibody (1 ⁇ g / ml PBS / 0.5% BSA) are used as a positive control.
• the mixture is then incubated with shaking at room temperature for 2 hours and finally washed with 3 ⁇ 350 ⁇ l 0.15 mol / l NaCl / 0.05% Tween 20.
• the GAD was obtained from pig brain using the method of Spink et al. (J. Neurochemistry, 40, 4, (1983) 1113).
• a fraction from the Sepharose S-200 gel filtration was used, in which the enzyme was present in approximately 90% purity.
• the enzyme was brought to a concentration of 10 ⁇ g / ml in coating buffer (Boehringer Mannheim GmbH, catalog No. 726559) and pipetted into Nunc Maxisorp immunoplates (50 ⁇ l / well). After an hour of incubation at 37 ° C., the coating solution was pipetted off and unspecific binding sites on the microtiter plate were saturated with 1% crotein C in PBS (200 ⁇ l / well; 1 hour at 37 ° C.).
• This mixture was added to the antigen-coated and washed wells of the microtiter plate and incubated at 4 ° C for two hours.
• Table III shows the results of the reaction of the monoclonal antibodies used with purified porcine GAD.

Landscapes

• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Medicinal Chemistry (AREA)
• Immunology (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Biochemistry (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Biophysics (AREA)
• Toxicology (AREA)
• Zoology (AREA)
• Gastroenterology & Hepatology (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Peptides Or Proteins (AREA)

Applications Claiming Priority (4)

Publications (3)

Family

ID=25901030

Family Applications (1)

Country Status (6)

Cited By (1)

Families Citing this family (17)

Family Cites Families (5)

• 1991
  • 1991-09-07 DE DE4129849A patent/DE4129849A1/de not_active Withdrawn
• 1992
  • 1992-02-10 EP EP92102187A patent/EP0499176B1/fr not_active Expired - Lifetime
  • 1992-02-10 AT AT92102187T patent/ATE161581T1/de not_active IP Right Cessation
  • 1992-02-10 DE DE59209079T patent/DE59209079D1/de not_active Expired - Fee Related
  • 1992-02-10 ES ES92102187T patent/ES2112869T3/es not_active Expired - Lifetime
  • 1992-02-14 JP JP4027942A patent/JPH07116239B2/ja not_active Expired - Fee Related
• 1995
  • 1995-11-07 US US08/554,796 patent/US5888813A/en not_active Expired - Fee Related
• 1998
  • 1998-07-29 US US09/124,410 patent/US6214568B1/en not_active Expired - Fee Related

Non-Patent Citations (6)

Also Published As

Similar Documents

Legal Events